Supplementary Materials for

www.advances.sciencemag.org/cgi/content/full/1/3/e1500183/dc1 Supplementary Materials for The microbiome of uncontacted Amerindians Jose C. Clemente, Erica C. Pehrsson, Martin J. Blaser, Kuldip Sandhu, Zhan Gao, Bin Wang, Magda Magris, Glida Hidalgo, Monica Contreras, Óscar Noya-Alarcón, Orlana Lander, Jeremy McDonald, Mike Cox, Jens Walter, Phaik Lyn Oh, Jean F. Ruiz, Selena Rodriguez, Nan Shen, Se Jin Song, Jessica Metcalf, Rob Knight, Gautam Dantas, M. Gloria Dominguez-Bello The PDF file includes: Published 17 April 2015, Sci. Adv. 1, e1500183 (2015) DOI: 10.1126/sciadv.1500183 Text Table S1. Samples obtained from the 34 Amerindian subjects included in this study: skin from right (BD) or left (BI) forearm (n = 28), oral (n = 28), and fecal (n = 12) specimens. Table S2. Composition of Amerindian E. coli isolate genomic libraries. Table S3. Functional capture of AR genes from Amerindian E. coli isolate genomic libraries. Table S4. AR genes identified from functional selections of Amerindian E. coli isolate genomic libraries and Amerindian and Puerto Rican metagenomic libraries. Table S5. Composition of metagenomic libraries from Amerindian and Puerto Rican fecal and oral microbiota. Table S6. Antibiotics used in genomic and metagenomic library selections for AR genes. Table S10. List of clusters at different k-mer and minimum contig length and their associated taxonomy. Table S11. Taxa of 329 bacterial strains cultured from feces of 12 isolated Amerindians. Table S12. Typing of 24 E. coli strains from 11 isolated Amerindians. Fig. S1. Meta-analysis of 16S V4 region fecal data. Fig. S2. Microbiome diversity between different human groups. Fig. S3. Microbiota diversity in fecal, oral, and skin samples from uncontacted Yanomami in relation to U.S. subjects using the V2 region of the 16S rrna gene. Fig. S4. Taxonomic distribution across gut samples sorted by the dominant genera within each population. Fig. S5. Prevalence/abundance curves of representative bacterial OTUs in feces (A), oral (B), and skin (C).

Fig. S6. ClonalFrame analysis of Escherichia coli strains based on the sequence of seven housekeeping genes. Fig. S7. PCoA plot of Yanomami, Malawian, and Guahibo fecal samples. Fig. S8. Fecal microbiome of non-bacteroides OTUs in Yanomami, Malawian, Guahibo, and U.S. samples. Fig. S9. Bacterial diversity as a function of age and body site. Fig. S10. Procrustes analyses of shotgun metagenomic and 16S rrna data for different subsets of samples. Fig. S11. Comparison of PICRUSt versus sequenced shotgun metagenomes of fecal and oral samples with at least 10,000 mapped reads. Fig. S12. Bacterial genome assembly. References (72 76) Other Supplementary Material for this manuscript includes the following: (available at www.advances.sciencemag.org/cgi/content/full/1/3/e1500183/dc1) Table S7 (Microsoft Excel format). List of all taxa significantly associated with each population in fecal samples as determined by LEfSe analysis. Table S8 (Microsoft Excel format). List of all taxa significantly associated with each population in oral samples as determined by LEfSe analysis. Table S9 (Microsoft Excel format). List of all taxa significantly associated with each population in skin samples as determined by LEfSe analysis.

Supplementary Materials 16S rrna sequences Quality-filtered, demultiplexed sequences from fecal samples were combined with those from a previous study that included a US population, and two semi-acculturated non-western populations 11. All sequences were trimmed at 100bp to avoid biases due to read length. We performed openreference OTU picking 72 at 97% identity to identify both characterized and novel taxa, and taxonomy was assigned using Greengenes version 13_8 73. Oral and skin samples were processed in a similar manner combined with data from the American Gut project instead. In fecal samples, we obtained 768,221 sequences for Yanomami subjects (69,838 + 6,630 seqs/sample), 913,44 for Guahibo (13,238 + 6,338), 820,776 for Malawians (15,199 + 3,152), and 4,510,511 for US (17,281 + 4,459). In oral samples, we obtained 1,820,607 sequences for Yanomami (67,429 + 16,553) and 890,367 for US (21,716 + 7,388). In skin samples, we obtained 1,516,780 sequences for Yanomami (54,170 + 9,571) and 872,475 for US (18,563 + 9,380). Since the oral and skin sites sampled in the Yanomami (buccal mucosa, volar forearm) were physically close but not identical to the sites sampled in US subjects (tongue, hand palm), we compared the distances per body site between the populations against those obtained from sub-body sites (i.e. buccal mucosa versus tongue, hand versus forearm) within populations. We found that the distances between populations for both oral and skin samples are significantly larger than distances between sub-body sites within a population (t-test, p < 0.01; Fig. S2F), indicating that our results are robust. Bacterial diversity in feces and skin, but not in the mouth, was significantly higher in Yanomami than in US subjects (Fig. 1A, 1E, 1I). Noticeably, fecal diversity was higher than in the semitransculturated Guahibo Amerindians (ANOVA and Tukey s HSD test p < 0.001; Fig. 1A). PCoA of UniFrac distances reveals that bacterial communities from each body site segregate Westernized people from agrarian and hunter-gatherer groups (Figs. 1B, 1F, 1J, and S1). Metagenome prediction and pathway analysis We used PICRUSt 1.0.0 18 with default parameters to predict metagenomic content from the 16S rrna data. KEGG Ortholog (KO) counts were thus assigned to each sample, and KEGG pathways annotated to each KO. STAMP 43 was then utilized in combination with ANOVA with the Tukey- Kramer post-hoc test and Bonferroni correction for multiple comparisons to detect pathways differentially abundant across populations. Effect sizes were measured using η 2 (eta-squared). Shotgun metagenomic sequencing and pre-processing We used the Illumina Nextera kit to perform shotgun metagenomic sequencing of 22 samples (8 fecal, 14 oral). 1 μl of metagenomic DNA from each sample was used as starting material. The dual-index multiplexed library was sequenced on an Illumina NextSeq (2x150bp). Shotgun reads were demultiplexed, allowing two mismatches per index, and trimmed with Trimmomatic v.0.30 74 in paired-end palindrome mode to remove Nextera adapter sequences and low quality (<Q13) bases from the ends (minimum length 36 bases). Computational filtering of assembled contigs from metagenomic libraries From the sequenced Amerindian MDA NTCs, 2321 contigs assembled. By comparison to NCBI nt (August 15, 2013), all but five contigs aligned most closely to Escherichia species (including one in which E. coli was the second hit); the remaining contigs align most closely to cloning vectors, Taq polymerase, or rabbit hemoglobin, all of which are commonly used molecular biology tools, trace amounts of which may have been present in the MDA reagents. Under our extremely stringent exclusion criteria (see Methods), most Amerindian metagenomic library-isolated genes from E. coli were excluded, as were many β-lactamases that aligned to a cloning vector on a NTC contig. The excluded contigs likely include some true AR genes present in

the Amerindian microbiota, but we gave exclusion of false positives greater priority in this investigation due to the unique nature of the samples. We were also able to recover some of the true positives excluded by NTC-masking by separately interrogating cultured E. coli isolates from the Amerindian individuals. Novel resistance gene with unknown function The AR genes isolated from the Amerindian metagenomic library selection include a previously unrecognized gene conferring resistance to chloramphenicol, O23_CH_19:550-804. The 255bp ORF is unannotated, and the top blastn alignments in NCBI nt are Neisseria lactamica plasmids pnl14 and pnl871104, which have no annotation for that region (blastn: 98%, DQ229164.1 and DQ229166.1). The ORF is situated on a 4398bp contig (KJ910975) that contains a number of mobile genetic elements, including a~1.4kb region spanning an adjacent relaxase/mobilization domain and mobc that aligns with 95% nucleotide identity to Neisseria gonorrhoeae plasmids (FJ172221.1, CP003910.1). Although this gene is represented in NCBI, its resistance function was previously unrecognized. Mobile genetic elements syntenic with antibiotic resistance genes Of the AR genes isolated with functional selections, several are syntenic with mobile genetic elements, including six Amerindian genes (four isolated from metagenomic libraries and two from E. coli isolate libraries) and two Puerto Rican genes. O23_CH_19 is discussed above. The chloramphenicol acetyltransferases on F6_CH_2 are nearly identical and flank a plasmid mobilization element whose closest homolog in NCBI nr is Oscillibacter sp. KLE 1728 (WP_021751621.1; 74% local identity). They share 99-100% nucleotide identity with HMP stool assemblies and a plasmid from the fish pathogen Aeromonas salmonicida (GI:500229267). The AraC transcriptional s on F23_CH_11, Library_A_CH_8, and Library_B_CH_15 have no hits in NCBI nt, but blastx to NCBI nr indicates they are rama homologs, and the top hit for all is E. coli. They are also adjacent to transposases that are 94-97% identical to E. coli (ref WP_032270577.1, ref WP_000343728.1, ref WP_021499094.1 ). Finally, the tetx variants on T1003_TE_12 and T1003_TG_1 are syntenic with integrases. The ribosomal protection protein on F6_TE_1 is not adjacent to a mobile element, but is widespread in industrialized settings. The tetw variant aligns to HMP assemblies from stool, oral cavity, nasal cavity, skin, and vaginal sites and with 99% nucleotide identity to 32 species in NCBI, including gut commensals from two phyla and Clostridium difficile (Fig. 3D). tetw is also widespread in animal gut microbiota and the environment 75 and is frequently associated with mobile genetic elements. Future research could determine whether the Yanomami genes associated with mobile elements were ubiquitous prior to the antibiotic era, or whether the new antibiotic selective pressure drove their dissemination 7 6. O23 CH 21 is also present in human pathogens, including Haemophilus influenzae, Klebsiella pneumoniae, and Vibrio spp. (Fig. 3C). The gene is often flanked by transposases that may contribute to its spread, although not in this context. Alignment of functionally selected Amerindian and Puerto Rican AR genes Seven of the Amerindian genes had homologs in fecal and oral metagenomic libraries from five Puerto Rican subjects, also identified by functional selections. Two pairs aligned with 95% nucleotide identity: the tetw variants from F6_TE_1 and 341_TE_1 and the Neisseria PBPs from O3_CZ_2_1/O3_CZ_2_2 and T1001_PITZ_5/T1001_CZ_11. Alignment of functionally selected AR genes to the Human Microbiome Project and MetaHIT All 108 AR genes were aligned to the HMP and MetaHIT assemblies. Excluding ribosomal protection proteins and tetracycline inactivation proteins, with a few exceptions, genes from the

fecal microbiota align only to HMP stool assemblies and MetaHIT, and genes from the oral microbiota align only to the HMP oral assemblies. The exceptions are genes from the E. coli isolates and F5_PITZ_3:33-794 (100% amino acid identical to ydeo from E. coli str. K-12 substr. MG1655), which align to HMP subgingival plaque assemblies with >95% nucleotide ID, representing potential assembly contamination; oral-isolated ABC transporter 337_TE_6:2-1678, which aligns to HMP stool with only 75.5% nucleotide ID; and two β-lactamases from oral library 238_PE_3, whose top hits in NCBI nr are Enterobacteriaceae, that align to MetaHIT.

Table S1. Samples obtained from the 34 Amerindian subjects included in this study: skin from right (BD) or left (BI) forearm (n=28), oral (n=28) and fecal (n=12) specimens. Subject ID Age (y) Gender Sampled sites Skin ORAL FECAL Left arm Right arm 24 4 m BI24 BD24 O24 20 7 f BI20 BD20 O20 23 7 f BI23 BD23 O23 H23 21 8 f BD21 O21 1 10 m BI1 BD1 O1 3 11 m BI3 BD3 O3 H3 11 11 m BI11 BD11 O11 9 12 f BI9 BD9 O9 H9 2 12 m BI2 BD2 O2 19 13 m BI19 BD19 O19 15 14 f BI15 BD15 18 17 f BI18 BD18 O18 31 17 m H31 29 18 m BD29 49 19 m BI49 BD49 O49 H49 51 19 m BI51 BD51 O51 5 20 m BI5 O5 H5 50 22 m BI50 BD50 O50 H50 22 23 f BI22 BD22 O22 36 23 m H36 26 30 m H26 25 35 f BI25 BD25 O25 4 36 m BI4 BD4 O4 48 37 m H48 41 39 m H41 8 40 m BI8 BD8 O8 10 40 m BI10 BD10 O10 12 45 f BI12 BD12 O12 16 45 f BI16 BD16 O16 17 46 m 2 BI17 BD17 O17 14 47 f BI14 BD14 O14 13 48 f BI13 BD13 O13 6 48 m BI6 O6 H6 7 50 m BI7 BD7 O7 54 - - BI54 BD54 O54

Table S2. Composition of Amerindian E. coli isolate genomic libraries. Subject IDs Strain IDs* Library size (GB) Library A 3, 5, 6, 23 15, 24, 30, 31, 55, 56, 122, 145, 188, 212, 4.50 4.95 217, 264, 293, 392, 393, 399, 402, 848, 849 Library B 9, 25, 31, 36, 48, 335, 373, 378, 383, 408, 428, 429, 431, 451, 9.04 49, 50 453, 478, 482, 502, 507, 525, 533, 564, 656, 1057 *Equal amounts of DNA were pooled from each isolate

Table S3. Functional capture of antibiotic resistance genes from Amerindian E. coli isolate genomic libraries. Antibiotic Library A Library B Penicillin ARG ARG Piperacillin* ARG ARG Piperacillin-tazobactam* ARG ARG Cefotaxime* NO-ARG NO-ARG Ceftazidime ARG ARG Cefepime* NO-ARG NO-ARG Meropenem* NO-ARG NO-ARG Aztreonam* ARG ARG Chloramphenicol ARG ARG Tetracycline* ARG ARG Tigecycline ARG ARG Gentamicin* NO-ARG NO-ARG Ciprofloxacin* NO-ARG NO-ARG Colistin NO-ARG NO-ARG ARG = Resistance (i.e., growth on selection plates in the absence of growth on negative control plates) was observed by functional screening of genomic library on corresponding antibiotic NO-ARG = No resistance was observed by functional screening of genomic library on corresponding antibiotic * All Amerindian E. coli isolates were screened for phenotypic resistance against these antibiotics, and were found to be susceptible in all cases. The composition of each library is defined in Table S2.

Table S4. Antibiotic resistance genes identified from functional selections of Amerindian E. coli isolate genomic libraries and Amerindian and Puerto Rican metagenomic libraries Origin Subject Site Contig Accession Gene MG1655 Length Top hit by blastx to Top hit Antibiotic Class %ID coordinates gene (bp) NCBI nr accession AE A Fecal Library A_PE_9 KJ910913 2948-4114 PE 446841495 ampc 1167 Escherichia coli* 98.7 Class C, ampc AE B Fecal Library B_PE_6 KJ910933 2782-3948 PE 446841455 ampc 1167 Escherichia coli* 99.2 Class C, ampc AE A Fecal Library A_PI_1 KJ910915 445-1611 PI 446841495 ampc 1167 Escherichia coli* 99.2 Class C, ampc AE B Fecal Library B_PI_8 KJ910938 3221-3490 PI β-lactamase ampc 270 Escherichia coli* 98.9 485961087 AE B Fecal Library B_PI_8 KJ910938 3465-4388 PI 446841495 ampc 924 Escherichia coli* 99 Class C, ampc AE B Fecal Library B_PI_10 KJ910936 2960-3229 PI β-lactamase ampc 270 Escherichia coli* 98.9 485961087 AE B Fecal Library B_PI_10 KJ910936 3204-4127 PI 446841495 ampc 924 Escherichia coli* 99 Class C, ampc AE A Fecal Library A_PITZ_1 KJ910914 1989-2996 PITZ 485706785 ampc 1008 Escherichia coli* 99.7 Class C, ampc AE B Fecal Library B_PITZ_8 KJ910934 1924-3090 PITZ 446841455 ampc 1167 Escherichia coli* 99.5 Class C, ampc AE A Fecal Library A_CZ_1 KJ910910 22-1188 CZ 446841447 ampc 1167 Escherichia coli* 100 Class C, ampc AE B Fecal Library B_CZ_1 KJ910932 78-1244 CZ 446841447 ampc 1167 Escherichia coli* 99.5 Class C, ampc AE A Fecal Library A_AZ_1 KJ910904 54-860 AZ β-lactamase ampc 807 Escherichia coli* 99.6 446841448 AE A Fecal Library A_AZ_1 KJ910904 847-1221 AZ β-lactamase ampc 375 Escherichia coli* 100 485961087 AE B Fecal Library B_AZ_1 KJ910926 161-1294 AZ 485828396 ampc 1134 Escherichia coli* 100 Class C, ampc AE A Fecal Library A_CH_10 KJ910905 2957-4189 CH MFS transporter 99.8 669336572 mdfa 1233 Enterobacteriaceae - 100 AE B Fecal Library B_CH_4 KJ910931 3012-4244 CH MFS transporter mdfa 1233 Escherichia coli ISC7 99.5 571185026 AE A Fecal Library A_TE_12 KJ910916 70-606 TE MFS transporter 491278665 bcr 537 Shigella sonnei* 100 bcr/cfla AE A Fecal Library A_TE_12 KJ910916 623-1279 TE MFS transporter 559165244 bcr 657 Escherichia coli* 99.5 bcr/cfla AE A Fecal Library A_TE_7 KJ910920 469-2250 TE ABC transporter mdlb 1782 Escherichia coli* 99.5 487358840 AE A Fecal Library A_TE_7 KJ910920 2243-4015 TE ABC transporter Escherichia coli 2-222- 652048337 mdla 1773 99.7 05_S1_C1 AE A Fecal Library A_CH_4 KJ910907 935-1324 CH AE B Fecal Library B_CH_10 KJ910927 2188-2577 CH AE A Fecal Library A_PE_6 KJ910912 3-308 PE transcriptional, mara transcriptional, mara transcriptional mara 390 mara 390 mara 306 Enterobacteriaceae* Enterobacteriaceae* Enterobacteriaceae* 100 100 100 445942659 445942659 445942659 AE A Fecal Library A_TE_4 KJ910917 1049-1438 TE mara 390 99.2 445942659

AE B Fecal Library B_TE_7 KJ910941 1067-1456 TE AE A Fecal Library A_TE_8 KJ910921 553-936 TE AE A Fecal Library A_TG_10 KJ910922 1093-1482 TG AE A Fecal Library A_CH_3 KJ910906 3520-4389 CH AE B Fecal Library B_CH_20 KJ910930 157-1026 CH AE A Fecal Library A_PE_2 KJ910911 631-1500 PE AE A Fecal Library A_TE_6 KJ910919 2033-2902 TE AE B Fecal Library B_TE_1 KJ910939 1855-2724 TE AE A Fecal Library A_TG_6 KP063058 2116-2985 TG AE A Fecal Library A_CH_6 KJ910908 476-799 CH AE B Fecal Library B_CH_17 KJ910929 139-462 CH AE A Fecal Library A_TE_5 KJ910918 3-299 TE AE B Fecal Library B_TE_6 KJ910940 1-198 TE AE A Fecal Library A_TG_1 KJ910923 2763-3086 TG AE A Fecal Library A_TG_4 KJ910924 515-838 TG transcriptional, mara transcriptional transcriptional, mara transcriptional, mara transcriptional transcriptional transcriptional transcriptional transcriptional transcriptional transcriptional transcriptional transcriptional transcriptional transcriptional transcriptional mara 390 mara 384 mara 390 rob 870 rob 870 rob 870 rob 870 rob 870 rob 870 soxs 324 Enterobacteriaceae* Enterobacteriaceae* Enterobacteriaceae* Enterobacteriaceae* Enterobacteriaceae* Enterobacteriaceae* Enterobacteriaceae* Enterobacteriaceae* Enterobacteriaceae* Enterobacteriaceae* Enterobacteriaceae* 96.9 99.2 99.2 100 100 100 100 100 99.7 100 soxs 324 Escherichia coli 99.1 soxs 297 Escherichia coli 100 soxs 198 Shigella dysenteriae* 100 soxs 324 Escherichia coli 99.1 soxs 324 Enterobacteriaceae* 99.1 445942659 445942659 445942659 446293811 446293811 446293811 446293811 446293811 446293811 445941503 585364975 585364975 445941496 585364975 445941503 AE A Fecal Library A_CH_8 KJ910909 1691-2029 CH n/a 339 Escherichia coli 99.1 607731705

transcriptional O121:H7 str. 2009C- 3299 Escherichia coli 607731705 AE B Fecal Library B_CH_15 KJ910928 4-387 CH transcriptional n/a 384 O121:H7 str. 2009C- 3299 91.5 446161253 AE B Fecal Library B_PI_1 KJ910937 3-560 PI transcriptional n/a 558 Escherichia coli* 100 446161253 AE B Fecal Library B_PITZ_9 KJ910935 1-690 PITZ transcriptional n/a 690 Escherichia coli* 100 446161253 AE A Fecal Library A_TG_8 KJ910925 1-564 TG transcriptional n/a 564 Escherichia coli* 99.5 AM 5 Fecal F5_PITZ_3 KJ910968 33-794 PITZ transcriptional - 762 Escherichia coli* 100 44598264 0 AM 6 Fecal F6_PI_27 KJ910949 1276-2184 PI 64755020 Bacteroides Class A, subclass - 909 95 1 stercorirosoris cbla AM 6 Fecal F6_CT_1 KJ910947 1200-2108 CT 64755020 Bacteroides Class A, subclass - 909 95.7 1 stercorirosoris cbla AM 6 Fecal F6_CZ_1 KJ910948 1330-2238 CZ 64755020 Bacteroides Class A, subclass - 909 94.7 1 stercorirosoris cbla AM 6 Fecal F6_CP_1 KJ910946 308-1216 CP 64755020 Bacteroides Class A, subclass - 909 95.7 1 stercorirosoris cbla AM 6 Fecal F6_AZ_9 KJ910942 1352-2260 AZ 64755020 Bacteroides Class A, subclass - 909 95.7 1 stercorirosoris cbla AM 3 Oral O3_CZ_2_1 KJ910979 301-2049 CZ Penicillin-binding 64754487-1749 Neisseria mucosa* 100 protein 1 AM 3 Oral O3_CZ_2_2 KJ910980 301-2049 CZ Penicillin-binding 64754487-1749 Neisseria mucosa* 99.8 protein 1 AM 5 Oral O5_CZ_1 KJ910969 522-2270 CZ Penicillin-binding 64754469-1749 Neisseria perflava 99.8 protein 5 AM 5 Oral O5_CZ_2 KJ910970 3-746 CZ Penicillin-binding 64754483-744 Neisseria mucosa 99.6 protein 5 AM 5 Oral O5_CZ_4 KJ910971 287-2062 CZ Penicillin-binding 64825514-1776 Kingella kingae 98 protein 1 AM 23 Oral O23_CZ_1 KJ910977 121-1902 CZ Penicillin-binding 48989383-1782 Kingella oralis* 80.8 protein 4 AM 3 Fecal F3_CH_11 KJ910965 1187-1852 CH Chloramphenicol uncultured bacterium 60224928-666 61.1 acetyltransferase DCM009Cm07 1 AM 5 Fecal F5_CH_2 KJ910967 271-807 CH Chloramphenicol Clostridium sp. 51441756-537 96.6 acetyltransferase CAG:226* 0 AM 6 Fecal F6_CH_2 KJ910943 1345-1971 CH Chloramphenicol Aeromonas 50022926-627 99.5 acetyltransferase salmonicida* 7 AM 6 Fecal F6_CH_2 KJ910943 2946-3572 CH Chloramphenicol - 627 Aeromonas 100 50022926

acetyltransferase salmonicida* 7 AM 6 Fecal F6_CH_4 KJ910944 174-1193 CH Chloramphenicol uncultured bacterium 60224928-1020 61.1 acetyltransferase DCM009Cm07 1 AM 6 Fecal F6_CH_5 KJ910945 198-752 CH Chloramphenicol 49390059-555 Prevotella copri* 90.4 acetyltransferase 2 AM 23 Fecal F23_CH_11 KJ910951 1298-1636 CH transcriptional - 339 Escherichia coli O121:H7 str. 2009C- 99.1 60773170 5 3299 AM 23 Oral O23_CH_21 KJ910976 997-1638 CH Chloramphenicol 50007534-642 100 acetyltransferase Gammaproteobacteria* 0 AM 23 Oral O23_CH_19 KJ910975 550-804 CH Unknown - 255 Neisseria bacilliformis* 61.3 49457489 5 AM 3 Fecal F3_TE_1 KJ910966 770-2674 TE Ribosomal [Clostridium] 50303880-1905 65.7 protection protein saccharolyticum* 5 AM 6 Fecal F6_TE_1 KJ910950 1651-3570 TE Ribosomal 48865001-1920 99.8 protection protein Bacteria* 5 AM 23 Fecal F23_TE_1 KJ910953 69-1403 TE MATE transporter - 1335 Phascolarctobacterium 49643688 99.1 succinatutens* 1 AM 3 Oral O3_TE_1 KJ910981 208-1638 TE MFS transporter 48987776-1431 Kingella denitrificans* 96.2 emrb 8 AM 5 Oral O5_TE_1 KJ910972 769-2208 TE MFS transporter 48985771-1440 Neisseria sicca* 95 emrb 7 AM 5 Oral O5_TE_3 KJ910973 2-703 TE MFS transporter - 702 Kingella denitrificans* 97.3 48987776 8 AM 23 Oral O23_TE_1 KJ910978 1-1845 TE ABC transporter - 1845 Cardiobacterium 49404210 97.1 valvarum* 2 PR 40 Fecal 220_PE_14 KJ911003 3-656 PE β-lactamase - 654 Collimonas 50377250 37 fungivorans* 3 PR 44 Oral 238_PE_3 KJ911002 446-805 PE 48767896-360 Escherichia coli* 98.3 Class A 6 PR 44 Oral 238_PE_3 KJ911002 777-1223 PE 34422264-447 Klebsiella pneumoniae 99.3 Class A 7 PR 62 Oral 337_PE_1 KJ910989 390-1307 PE 49182321-918 99 Class A Pasteurellaceae* 3 PR 62 Oral 337_PI_2 KJ910996 201-1118 PI 49182321-918 99 Class A Pasteurellaceae* 3 PR 62 Oral 337_PITZ_2 KJ910995 282-1199 PITZ 49182321-918 99.3 Class A Pasteurellaceae* 3 PR 62 Oral 337_CZ_1 KJ910983 703-1620 CZ 49182321-918 100 Class A Pasteurellaceae* 3 PR 62 Oral 337_PE_13 KJ910985 5065-5652 PE Class B, subclass - 588 uncultured bacterium 99.5 63666984 6 B3 PR 62 Oral 337_PE_15 KJ910987 116-1045 PE Class B, subclass - 930 uncultured bacterium 100 63666984 6 B3 PR 62 Oral 337_PE_16 KJ910988 1046-1969 PE Class B, subclass - 924 uncultured bacterium 99.7 63666932 5 B3 PR 62 Oral 337_PE_24 KJ910991 837-1709 PE - 873 uncultured bacterium* 100 63666984

PR 62 Oral 337_PE_30 KJ910992 3280-3897 PE PR 62 Oral 337_PE_34 KJ910993 562-1008 PE PR 62 Oral 337_PE_11 KJ910984 464-1354 PE PR 62 Oral 337_PE_14 KJ910986 712-1602 PE PR 62 Oral 337_PE_34 KJ910993 1005-1445 PE PR 62 Oral 337_PE_3 KJ910994 1205-2056 PE PR 62 Oral 337_PE_13 KJ910985 4244-4666 PE Class B, subclass B3 Class B, subclass B3 Class B, subclass B3 Class B, subclass B3, BJP Class B, subclass B3, BJP Class B, subclass B3, BJP Class B, subclass B1, SPM Penicillin-binding protein PR T100 Oral T1001_PITZ_7 KJ910963 2-862 PITZ β-lactamase - 861-618 uncultured bacterium 98.5-447 uncultured bacterium 91.3-891 uncultured bacterium 100-891 uncultured bacterium 99.3-441 uncultured bacterium 100-852 uncultured bacterium 99.3-423 uncultured bacterium 98.6 Ktedonobacter racemifer* PR T100 Oral T1001_CZ_5 KJ910961 1-1212 CZ β-lactamase - 1212 Methanosarcina mazei* 38.3 PR T100 Oral T1001_PITZ_5 KJ910962 66-1814 PITZ PR T100 Oral T1001_CZ_11 KJ910959 1532-3334 CZ Penicillin-binding protein Penicillin-binding protein 46.4-1749 Neisseria macacae* 100-1803 Neisseria macacae* 99.6 PR 62 Oral 337_CH_1 KJ910982 349-1578 CH MFS transporter - 1230 Acinetobacter lwoffii 98.8 PR 40 Fecal 220_TE_2 KJ911006 6-1925 TE PR 40 Fecal 220_TE_6 KJ911008 118-2043 TE Ribosomal protection protein Ribosomal protection protein - 1920-1926 PR 40 Fecal 220_TE_1 KJ911005 74-1234 TE TetX - 1161 PR 44 Fecal 242_TE_1 KJ911009 523-1683 TE TetX - 1161 PR 44 Fecal 242_TG_2 KJ911011 209-1369 TG TetX - 1161 PR 62 Fecal 341_TE_1 KJ911000 202-2121 TE PR 62 Fecal 341_TE_2 KJ911001 837-1877 TE Ribosomal protection protein Ribosomal protection protein - 1920-1041 Firmicutes** Prevotella* Bacteroidales** Bacteroides fragilis str. S23L17* Bacteroidales* Bacteria** Bacteria** PR T100 Fecal T1003_TE_12 KJ910954 588-1748 TE TetX - 1161 Bacteroides fragilis* 99.7 PR T100 Fecal T1003_TG_1 KJ910958 1021-2181 TG TetX - 1161 Bacteroides fragilis* 99.7 99.7-99.8 100 99.2 99.5 99.7 99.8-100 98.8-100 2 63666950 8 63666934 3 63666951 2 63666983 0 63666934 3 63666952 0 63666983 5 49519852 1 49934551 6 48987269 1 48987269 1 49801347 4 64957194 7 49050564 9 59886750 7 59886750 7 49592650 3 35666863 3 44661439 0 49223286 5 49223286 5

PR 62 Oral 337_TE_6 KJ910999 2-1678 TE ABC transporter - 1677 Neisseria cinerea* 99.8 48977306 7 PR 62 Oral 337_TE_2 KJ910997 82-1290 TE MFS transporter 48977278-1209 Neisseria cinerea* 98.5 bcr/cfla 7 PR 62 Oral 337_TE_4 KJ910998 156-1589 TE MFS transporter 48986319-1434 Neisseria sicca* 98.7 emrb 9 PR T100 Oral T1001_TE_1 KJ910964 166-2085 TE Ribosomal 44661441-1920 100 protection protein Bacteria* 2 AE: Amerindian E. coli isolate, AM: Amerindian microbiota, PR: Puerto Rican microbiota. A and B are libraries created from the pooled genomic DNA of Amerindian E. coli isolates (Table S2). PE: penicillin, PI: piperacillin, PITZ: piperacillin-tazobactam, CT: cefotaxime, CZ: ceftazidime, CP: cefepime, AZ: aztreonam, CH: chloramphenicol, TE: tetracycline, TG: tigecycline. MG1655 gene was identified through alignment to Escherichia coli str. K-12 substr. MG1655; all alignments 96% nucleotide ID. Investigated for E. coli isolate libraries only. * The top hit comprises multiple accessions, and only the first is listed. **The gene aligned equally well to multiple accessions, and only one is listed.

Table S5. Composition of metagenomic libraries from Amerindian and Puerto Rican fecal and oral microbiota. Origin Individual Sex Age Site ID Library size (GB) Amerindian 3 M 11 Fecal F3 7.15 Oral O3 7.48 Amerindian 5 M 20 Fecal F5 0.80 Oral O5 8.69 Amerindian 6 M 48 Fecal F6 4.80 Oral O6 0.49 Amerindian 23 F 7 Fecal F23 3.19 Oral O23 3.00 Puerto Rican 40 M 9 Fecal 220 1.08 Puerto Rican 62 M 13 Oral 337 4.09 Fecal 341 5.77 Puerto Rican 70 M 20 Oral 381 1.41 Puerto Rican T100 M 45 Oral T100-1 8.51 Fecal T100-3 4.32 Puerto Rican 44 F 7 Oral 238 1.75 Fecal 242 6.44

Table S6. Antibiotics used in genomic and metagenomic library selections for antibiotic resistance genes. Cellular target Antibiotic Abbreviation Source Concentration (ug/ml) Cell wall Penicillin PE Natural 128 synthesis Piperacillin PI Semi-synthetic 16 Piperacillintazobactam PITZ Semi-synthetic 16/4 Cefotaxime CT Semi-synthetic 8 Ceftazidime CZ Semi-synthetic 16 Cefepime CP Semi-synthetic 8 Meropenem ME Semi-synthetic 16 Aztreonam AZ Synthetic 8 Protein synthesis Chloramphenicol CH Natural 8 Tetracycline TE Natural 8 Tigecycline TG Semi-synthetic 2 Gentamicin GE Natural 16 DNA replication Ciprofloxacin CI Synthetic 0.5 Cell membrane Colistin CL Natural 8 Other Nitrofurantoin NI Synthetic 32

Table S10. List of clusters at different k-mer and minimum contig length and their associated taxonomy. cluster k- mer size threshold length taxonomy 0 55 100 unidentified 1 55 100 unidentified 2 55 100 unidentified 3 55 100 unidentified 4 55 100 Mollicutes_bacterium Fusobacterium_ulcerans 5 55 100 unidentified 6 55 100 unidentified 7 55 100 Candidatus_Pelagibacter_ubique_SAR11 8 55 100 unidentified 9 55 100 Prevotella_sp._Oral_Taxon_472 Prevotella_ruminicola Prevotella_copri Prevotella_melaninogenica 10 55 100 Prevotella_pallens_Oral_Taxon_714 Prevotella_melaninogenica Prevotella_denticola 11 55 100 unidentified 12 55 100 unidentified 13 55 100 Prevotella_copri 14 55 100 unidentified 15 55 100 unidentified 16 55 100 unidentified 17 55 100 unidentified 18 55 100 Beggiatoa_sp. 19 55 100 Mollicutes_bacterium 20 55 100 unidentified 21 55 100 Prevotella_denticola Prevotella_melaninogenica 22 55 100 unidentified 23 55 100 Streptococcus_mitis 24 55 100 unidentified cluster k- mer size threshold length taxonomy 0 59 100 unidentified 1 59 100 Prevotella_melaninogenica Prevotella_denticola Prevotella_pallens_Oral_Taxon_714 2 59 100 Granulicatella_elegans 3 59 100 unidentified 4 59 100 unidentified 5 59 100 Streptococcus_sanguinis

Streptococcus_oralis Streptococcus_mitis Streptococcus_sp. Streptococcus_pneumoniae Veillonella_atypica 6 59 100 unidentified 7 59 100 unidentified 8 59 100 unidentified 9 59 100 unidentified 10 59 100 unidentified 11 59 100 unidentified 12 59 100 unidentified 13 59 100 unidentified 14 59 100 Prevotella_copri 15 59 100 Streptococcus_sanguinis Streptococcus_oralis Streptococcus_sp. Streptococcus_pneumoniae Streptococcus_infantis 16 59 100 unidentified 17 59 100 unidentified 18 59 100 Burkholderia_mallei 19 59 100 unidentified 20 59 100 unidentified 21 59 100 unidentified 22 59 100 Streptococcus_sanguinis Streptococcus_dysgalactiae_equisimilis Streptococcus_sp. Streptococcus_pneumoniae Streptococcus_infantis 23 59 100 unidentified 24 59 100 unidentified 25 59 100 Fusobacterium_ulcerans Mollicutes_bacterium 26 59 100 unidentified 27 59 100 Streptococcus_mitis cluster k- mer size threshold length taxonomy 0 59 200 Granulicatella_elegans 1 59 200 unidentified 2 59 200 unidentified 3 59 200 Streptococcus_mitis 4 59 200 Neisseria_elongata_glycolytica

Neisseria_mucosa Neisseria_subflava Neisseria_macacae Neisseria_sp._oral_taxon_014 Neisseria_gonorrhoeae Neisseria_flavescens 5 59 200 unidentified 6 59 200 unidentified 7 59 200 unidentified 8 59 200 unidentified 9 59 200 Neisseria_lactamica Neisseria_elongata_glycolytica Neisseria_sicca Neisseria_mucosa Neisseria_subflava Neisseria_macacae Neisseria_meningitidis Neisseria_sp._oral_taxon_014 Neisseria_cinerea Neisseria_gonorrhoeae Neisseria_flavescens 10 59 200 Prevotella_copri 11 59 200 Neisseria_mucosa 12 59 200 Prevotella_marshii Prevotella_melaninogenica Bacteroides_xylanisolvens Prevotella_ruminicola Chryseobacterium_gleum Zunongwangia_profunda Prevotella_sp._Oral_Taxon_472 Bacteroides_intestinalis Bacteroides_uniformis Parabacteroides_johnsonii Bacteroides_cellulosilyticus Bacteroides_sp. Prevotella_buccalis Bacteroides_coprophilus Bacteroides_eggerthii Bacteroides_finegoldii Bacteroides_thetaiotaomicron Prevotella_buccae Alistipes_shahii

Prevotella_sp._Oral_Taxon_299 Riemerella_anatipestife Flavobacteriacea_bacterium Prevotella_timonensis Parabacteroides_sp. Bacteroides_stercoris Gramella_forsetii Bacteroides_dorei 13 59 200 unidentified 14 59 200 unidentified 15 59 200 unidentified 16 59 200 Fusobacterium_ulcerans Mollicutes_bacterium 17 59 200 unidentified 18 59 200 unidentified 19 59 200 Neisseria_sicca Neisseria_mucosa 20 59 200 unidentified 21 59 200 unidentified 22 59 200 Neisseria_meningitidis Neisseria_mucosa Kingella_denitrificans_Oral_Taxon_582 Neisseria_sicca Neisseria_elongata_glycolytica 23 59 200 Lactobacillus_helveticus Streptococcus_sanguinis Streptococcus_gordonii Streptococcus_oralis Streptococcus_sp._Oral_Taxon_071 Streptococcus_parasanguinis Streptococcus_sp. Streptococcus_salivarius Streptococcus_infantis 24 59 200 Streptococcus_sp. 25 59 200 Neisseria_mucosa 26 59 200 unidentified cluster k- mer size threshold length taxonomy 0 59 500 Lysinibacillus_fusiformis Bacillus_thuringiensis Streptococcus_pyogenes Enterococcus_casseliflavus_Oral_Taxon_801 Lactobacillus_gasseri

Streptococcus_sp. Streptococcus_uberis Lactobacillus_coryniformis_coryniformis Streptococcus_mutans Lactobacillus_fermentum Lactobacillus_antri Staphylococcus_capitis Streptococcus_sanguinis Streptococcus_pneumoniae Granulicatella_adiacens Bacillus_coahuilensis Granulicatella_elegans Streptococcus_equinus Bacillus_amyloliquefaciens_plantarum Enterococcus_italicus_Oral_Taxon_803 Streptococcus_oralis Lactobacillus_farciminis Lactobacillus_ruminis Streptococcus_dysgalactiae_equisimilis Streptococcus_peroris_Oral_Taxon_728 Enterococcus_faecium Eremococcus_coleocola Leuconostoc_mesenteroides_cremoris Aerococcus_viridans Staphylococcus_aureus Lactobacillus_coleohominis Streptococcus_anginosus Enterococcus_faecalis Lactobacillus_iners Enterococcus_gallinarum Listeria_grayi Streptococcus_sp._Oral_Taxon_07 Streptococcus_mitis Lactobacillus_coryniformis_torquens Staphylococcus_epidermidis Lactobacillus_amylolyticus Lactobacillus_reuteri Lactobacillus_fructivorans Weissella_paramesenteroides Streptococcus_thermophilus Anoxybacillus_flavithermus Lactobacillus_animalis

Streptococcus_infantis Streptococcus_salivarius Pediococcus_acidilactici Lactobacillus_buchneri Bacillus_subtilis_spizizenii Streptococcus_parasanguinis Bacillus_cereus Lactococcus_lactis_lactis Lactobacillus_salivarius Lactobacillus_johnsonii Lactobacillus_acidophilus Streptococcus_gordonii Streptococcus_suis Lactobacillus_rhamnosus Lactobacillus_delbrueckii_bulgaricus Bacillus_subtilis_subtilis Enterococcus_casseliflavus Gemmata_obscuriglobus Staphylococcus_aureus_aureus Streptococcus_australis Lactobacillus_paracasei 1 59 500 unidentified 2 59 500 Neisseria_mucosa Neisseria_flavescens Kingella_denitrificans_Oral_Taxon_582 Neisseria_sicca Neisseria_elongata_glycolytica Neisseria_meningitidis cluster k- mer size threshold length taxonomy 0 63 500 unidentified 1 63 500 Streptococcus_sp. Streptococcus_mutans Streptococcus_sanguinis Streptococcus_pneumoniae Streptococcus_equinus Streptococcus_infantis Streptococcus_salivarius Streptococcus_oralis Streptococcus_dysgalactiae_equisimilis Streptococcus_peroris_Oral_Taxon_728 Lactococcus_lactis_lactis Streptococcus_gordonii

Streptococcus_anginosus 2 63 500 unidentified 3 63 500 unidentified 4 63 500 Fusobacterium_nucleatum_vincentii Fusobacterium_gonidiaformans Fusobacterium_sp. 5 63 500 unidentified cluster k- mer size threshold length taxonomy 0 67 500 Prevotella_copri 1 67 500 Streptococcus_sanguinis Granulicatella_elegans Streptococcus_equinus Streptococcus_oralis Streptococcus_dysgalactiae_equisimilis Streptococcus_peroris_Oral_Taxon_728 Enterococcus_faecium Enterococcus_gallinarum Streptococcus_mitis Streptococcus_salivarius Lactococcus_lactis_lactis Streptococcus_australis Streptococcus_pyogenes Enterococcus_casseliflavus_Oral_Taxon_801 Streptococcus_sp. Streptococcus_mutans Streptococcus_pneumonia Granulicatella_adiacens Streptococcus_anginosus Enterococcus_faecalis Streptococcus_cristatus Lactobacillus_animalis Streptococcus_infantis Streptococcus_gordonii Streptococcus_suis 2 67 500 unidentified 3 67 500 Fusobacterium_sp. Fusobacterium_nucleatum_nucleatum Fusobacterium_nucleatum_vincentii Fusobacterium_gonidiaformans 4 67 500 unidentified 5 67 500 unidentified

Table S11. Taxa of 329 bacterial strains cultured from feces of 12 isolated Amerindians. Phylum Order Genus Species Subjects Isolates Actinobacteria Actinomycetales Arthrobacter sp a 1 1 Bacteroidetes Bacteroidales Bacteroides dorei 1 3 Bacteroidetes Bacteroidales Bacteroides eggerthii 1 1 Bacteroidetes Bacteroidales Bacteroides ovatus 2 2 Bacteroidetes Bacteroidales Porphyromonas sp a 1 2 Bacteroidetes Bacteroidales Porphyromonas sp a 1 1 Bacteroidetes Bacteroidales Prevotella sp a 1 1 Bacteroidetes Flavobacteria Capnocytophaga ochracea 1 1 Bacteroidetes Flavobacteria Capnocytophaga sp a 1 2 Bacteroidetes Flavobacteria Capnocytophaga sputigena 1 3 Firmicutes Bacillales Bacillus altitudinis 1 1 Firmicutes Bacillales Bacillus sp a 1 3 Firmicutes Clostridiales Clostridium clostridioforme 5 8 Firmicutes Clostridiales Clostridium perfringens 9 22 Firmicutes Clostridiales Clostridium sp a 8 17 Firmicutes Clostridiales Peptostreptococcus asaccharolyticus 3 4 Firmicutes Clostridiales Peptostreptococcus sp a 5 7 Firmicutes Lactobacillales Enterococcus durans 2 3 Firmicutes Lactobacillales Enterococcus faecium 1 1 Firmicutes Lactobacillales Enterococcus fecalis 2 12 Firmicutes Lactobacillales Enterococcus hirae 5 61 Firmicutes Lactobacillales Lactobacillus salivarius 1 1 Firmicutes Lactobacillales Lactococcus garvieae 6 29 Fusobacteria Fusobacteriales Fusobacterium sp a 1 1 Proteobacteria Enterobacteriales Escherichia coli 11 131 Proteobacteria Enterobacteriales Klebsiella pneumoniae 3 10 Proteobacteria Neisseriales Neisseria mucosa 2 1 a Only defined to genus level

Table S12. Typing of 24 E. coli strains from 11 isolated Amerindians Host ID Age Gender # Strain Sequence type MLST Cluster Clonal Complex Closest Reference Strain* Reference strain host Reference strain host species 23 7 Female 55 3314 A 10 Ecor16 Animal Captive leopard 23 7 Female 56 3315 A 10 Ecor10 + Ecor2 Human Human 23 7 Female 145 94 B1 58 Ecor45 Animal Domesticated pig 23 7 Female 402 94 B1 58 Ecor45 Animal Domesticated pig 3 11 Male 212 3322 B1 641 Ecor69 Animal Captive celebes ape 3 11 Male 217 3316 B1 Singleton Ecor29 Animal Wild kangaroo rat 3 11 Male 264 3316 B1 Singleton Ecor29 Animal Wild kangaroo rat 3 11 Male 392 3411 B1 641 Ecor69 Animal Captive celebes ape 3 11 Male 848 3316 B1 Singleton Ecor29 Animal Wild kangaroo rat 9 12 Female 373 94 B1 58 Ecor45 Animal Domesticated pig 31 17 Male 428 164 B1 Singleton Ecor34 Animal Domesticated dog Various Captive celebes ape + 31 17 Male 429 3318 A 361 (Ecor18,19,2 Animal Domesticated bovine 0,21) 5 20 Male 24 3313 A 10 Ecor16 Animal Captive leopard 5 20 Male 31 871 A 10 Ecor16 Animal Captive leopard 5 20 Male 122 94 B1 58 Ecor45 Animal Domesticated pig 5 20 Male 393 46 A 10 Ecor16 Animal Captive leopard Various 7 Human + 1 Human + Domesticated 50 22 Male 533 10 A 10 (Ecor14,12,1 Animal dog 1,1,9,8,5,25) 36 23 Male 564 3415 B1 Singleton Ecor27 Animal Captive giraffe 26 30 Male 335 656 A 10 Ecor6 Human Human 26 30 Male 383 115 D 115 Ecor46 Animal Captive celebes ape 26 30 Male 507 3414 B1 58 Ecor45 Animal Domesticated pig 48 37 Male 502 3413 B1 278 Ecor27 Animal Captive giraffe 6 48 Male 293 3317 A 10 Ecor15 Human Human 6 48 Male 399 3412 B1 278 Ecor27 Animal Captive giraffe

Lifestyle PC2-6% Westernized not Pseudo F = 66.9 p < 0.001 Lifestyle PC2-6% Population/country Westernized agrarian hunter-gatherer Pseudo F = 36.7 p < 0.001 USA Italy Malawi PC2-6% Tanzania Venezuela (Guahibo) Venezuela (Yanomami) Pseudo F = 18.5 p < 0.001 PC1-16% PC3-3% PC1-16% PC3-3% Supervised Learning on Lifestyle Agrarian versus hunter-gatherer PC2-4% PC1-16% PC3-3% agrarian (Malawi) agrarian (Venezuela) hunter-gatherer (Tanzania) hunter-gatherer (Venezuela) Westernized not westernized 270 4 agrarian agrarian 119 hunter-gatherer 11 Westernized 0 not 0 135 hunter-gatherer 0 4 0 Class error 0.0288 0 western 4 1 270 Class error 0.0325 0.75 0 PC1-6% PC3-4% Figure S1. Meta-analysis of 16S V4 region fecal data. PCoA plots based on unweighted UniFrac distances colored by A) whether the population was Westernized or not, B) by subsistence mode, C) by country, and D) agrarian versus hunter-gatherer. D) Pseudo-F and p-values for permanova tests for each category in A-C. Rarefied at 1,000 seqs per sample. Data are from Schnorr et al (2014) and Yatsunenko et al (2012). Lifestyle effect is much bigger than study effect (in PC2).

A B 0.9 UniFrac distances 0.8 250 4000 150 100 0.6 3000 0.5 Yanomami Malawi 50 75 100 0 # Individuals 25 50 75 0.7 0.6 Yanomami 25 US 0.8 0.8 Guahibo 0 0 Malawi Gut Group 1000 USA 50 Guahibo 2000 UniFrac distances mean # of bacterial genera 200 UniFrac distances mean PSR (phylogenetic species richness) 0.7 100 # Individuals 0.6 0.4 Yanomami C D Oral US Yanomami US Skin Bray Curtis distance 0.3 0.2 UniFrac distances 0.7 group between within 0.1 0.6 USA_Malawi USA_Guahibo USA_Yanomami Malawi_Guahibo Yanomami_Malawi Yanomami_Guahibo 0.5 0.6 group between within Bray Curtis distance Bray Curtis distance 0.4 0.4 0.5 0.3 group between within 0.2 0.2 0.1 Coromoto Platanillal Village Yanomami Yanomami_US Yanomami_US F PICRUSt accuracy (Spearman, r) E US 0.80 Unweighted UniFrac distances 0.8 Guahibo 0.6 0.4 0.75 0.2 Mucosa (Yanomami) vs tongue (US) 0.10 0.15 NSTI 0.20 Tongue vs mucosa, US HMP 0.9 Unweighted UniFrac distances 0.05 Tongue vs mucosa, US Costello et al 0.8 0.7 0.6 0.5 Hand (US) vs forearm (Yanomami) Hand vs forearm, US Costello et al

Figure S 2. Microbiome diversity between different human groups. (A) Gamma diversity curves for fecal samples. (Left) Mean number of bacterial genera as a function of number of samples. All differences significant (Welch s t-test with n = 10 subjects, FDR corrected p-values) (Right) Mean phylogenetic species richness as a function of number of samples. Non-western (Yanomami, Guahibo, Malawians) versus US differences significant (Welch s t-test with n = 10 subjects, FDR corrected p-values) (B) Unweighted UniFrac distances within population and body site. All differences were significant (p < 0.001, PERMANOVA with 999 permutations) (C) Unweighted UniFrac distances of fecal microbiota within villages in two Amerindian populations. Distances within samples from the uncontacted Yanomami village are significantly smaller than distances within either of the Guahibo villages, Coromoto and Platanillal (p < 0.001, PERMANOVA with 999 permutations) (D) Within versus between Bray-Curtis distances between all pairs of populations with metagenomic data. Separation between populations across body sites after randomly sub-sampling all populations to the same number of samples. Top: Fecal (p < 0.001, PERMANOVA with 999 permutations). Bottom, left: oral (p = 0.002, PERMANOVA with 999 permutations). Bottom, right: skin (p < 0.001, PERMANOVA with 999 permutations) (E) Correlation between shotgun- and PICRUSt-predicted metagenomes in a US and Guahibo population. Correlation measured as Spearman s r (vertical axis) versus mean closest reference genome measure as Nearest Sequenced Taxon Index (NSTI, horizontal axis). Mean NSTI of US subjects is not significantly lower than that of Guahibos (p = 0.1586, t-test), but Spearman s r is (p = 0.003, t-test) (F) Unweighted UniFrac distances between microbiota in different sub-sites of the same body location. (Top) Comparison between oral sub-sites. Left (this study): US oral mucosa vs Yanomami tongue; middle (Costello et al, Science 2009): US oral mucosa vs US tongue; right (HMP, Nature 2012): US oral mucosa vs US tongue. Differences between populations are larger than between sub-body sites (p < 0.001, PERMANOVA with 999 permutations) (Bottom) Comparison between cutaneous sub-sites. Left (this study): US hand vs Yanomami forearm; right (Costello et al, Science 2009): US hand vs US forearm. Differences between populations larger than between sub-sites (p < 0.001, PERMANOVA with 999 permutations)

Figure S3. Microbiota diversity in fecal, oral, and skin samples from uncontacted Yanomami in relation to US subjects using the V2 region of the 16S rrna gene. (A) Faith s phylogenetic diversity (average ± standard deviation) of fecal samples from Yanomami and US subjects. OTU tables rarefied at 10,000 sequences/sample. Interpopulation differences were significant (p < 1.23e-06, t-test) (B) PCoA plot based on UniFrac distances calculated on the OTU table of fecal samples rarefied at 10,000 sequences/sample. (C) Top discriminative bacteria among populations in fecal samples as determined by LEfSe analysis. (D) Normalized prevalence/abundance curves for all OTUs in fecal samples. (E) Faith s phylogenetic diversity (average ± standard deviation) of oral samples from Yanomami and US subjects. OTU tables rarefied at 10,000 sequences/sample. Inter-population differences were significant (p = 0.0001, t-test). (F) PCoA plot based on UniFrac distances calculated on OTU tables of oral samples rarefied at 10,000 sequences/sample. (G) Top discriminative bacteria among populations in oral samples as determined by LEfSe analysis. (H) Normalized prevalence/abundance curves for all OTUs in oral samples. (I) Faith s phylogenetic diversity (average ± standard deviation) of skin samples from Yanomami and US subjects. OTU tables rarefied at 1,000 sequences/sample. Inter-population differences were significant (p < 1.418e-13, t- test). (F) PCoA plot based on UniFrac distances calculated on OTU tables of skin samples rarefied at 1,000 sequences/sample. (G) Top discriminative bacteria among populations in skin samples as determined by LEfSe analysis. (H) Normalized prevalence/abundance curves for all OTUs in skin samples.

Figure S4. Taxonomic distribution across gut samples sorted by the dominant genera within each population. (A) From top to bottom, and left to right: Taxonomic abundance in fecal samples of US, dominated by Bacteroides; Malawi, Guahibo, and Yanomami, dominated by Prevotella. (B) Left to right: Taxonomic abundance in oral samples of US, dominated by Streptococcus and with significant abundance of Rothia and Yanomami, showing more even diversity. (C) Left to right: Taxonomic abundance in skin samples of US, dominated by Corynebacterium; Yanomami, showing more even diversity but also significant levels of Kocuria.

293. Figure S5. Prevalence/abundance curves of representative bacterial OTUs in feces (A), oral (B) and skin (C). Shaded areas encompass the mean and maximum curves generated over 10 independent rarefactions of the OTU table. The Greengenes OTU identifier is indicated in parentheses. Ecor48_Human Ecor50_Human Ecor49_Human Ecor35_Human Ecor36Human Ecor43_Human Ecor41_Human Ecor40_Human Ecor39_Human Ecor38_Human Ecor51_Human Ecor52_Orang utan Ecor44_Cougar Ecor47_Sheep Ecor46_Celebese ape 383. Ecor54_Human Ecor56_Human Ecor57_Gorilla 502. Ecor55_Human 564. Ecor53_Human Ecor60_Human Ecor59_Human Ecor27_Giraffe Ecor26_Human 399. Ecor61_Human Ecor62_Human Ecor69_Celebese ape 212. Ecor63_Human 392. Ecor66_Celebese ape Ecor37_Marmoset Ecor64_Human Ecor42_Human Ecor65_Celebese ape Ecor23_Elephant Ecor6_Human Ecor24_Human Ecor31_Leopard 335. 0.1 Ecor15_Human Ecor30_Bison Ecor32_Giraffe Ecor4_Human Ecor33_Sheep Ecor17_Pig 217. Ecor29_Kangaroo rat Ecor22_Bovine 264. 393. Ecor16_Leopard 848. 55. 31. 122. 145. Ecor68_Giraffe 24. 373. 402. 507. Ecor70_Gorilla Ecor71_Human Ecor72_Human 428. Ecor34_Dog Ecor67_Goat Ecor28_Human Ecor45_Pig Ecor58_Lion 56. 429. 533. Ecor1_Human Ecor10_Human Ecor11_Human Ecor12_Human Ecor13_Human Ecor14_Human Ecor2_Human Ecor25_Dog Ecor3_Dog Ecor5_Human Ecor8_Human Ecor9_Human Ecor18_Celebese ape Ecor19_Celebese ape Ecor20_Bovine Ecor21_Bovine Ecor7_Orang utan Human (Amerindian) Human (Westernized) Animal Figure S6. ClonalFrame analysis of Escherichia coli strains based on the sequence of 7 housekeeping genes. Strains included 72 reference collection Escherichia coli (ECOR) (circles, strains isolated from animals; squares, strains isolated from westernized humans) and 24 Amerindian isolates (triangles), the tree is rooted on Escherichia fergusonii (not shown). Non-pathogenic strains are indicated by a full symbol, whereas pathogenic strains are represented by an open symbol. Colors indicate the 5 major phylogenetic groups (blue, A; green, B1; red, B2; yellow, D; purple, E). For presentation purposes Amerindian isolates are indicated by black font, whereas the reference strains (ECOR) are indicated by grey font.

Yanomami Guahibo Malawi Figure S7. PCoA plot of Yanomami, Malawian, and Guahibo fecal samples. PCoA based on unweighted UniFrac distances on a table rarefied at 5,000 sequences per sample.

Figure S8. Fecal microbiome of non-bacteroides OTUs in Yanomami, Malawian, Guahibo, and US samples. (A) PCoA plot based on unweighted UniFrac distances on a table rarefied at 1,000 sequences per sample. (B) Faith s phylogenetic diversity (average ± standard deviation) of oral samples from Yanomami and US subjects. OTU table rarefied at 1,000 sequences per sample. A 50 USA Malawi B 25 US Yanomami 40 30 Phylogenetic diversity 20 15 10 R 2 = 0.0377 R 2 = 0.282 population Phylogenetic diversity 20 50 R 2 = 0.00581 R 2 = 0.277 Yanomami Guahibo C 5 40 20 40 60 80 20 40 60 80 Age US Yanomami US Yanomami 40 30 population USA Malawi Phylogenetic diversity 30 20 20 R 2 = 0.0482 R 2 = 0.0419 Yanomami Guahibo 10 R 2 = 0.0019 R 2 = 0.0206 population US Yanomami 20 40 60 80 20 40 60 80 Age 20 40 60 20 40 60 Age Figure S9. Bacterial diversity as a function of age and body site. (A) Average Faith s phylogenetic diversity of fecal samples from US, Malawi, Yanomami, and Guahibo subjects plotted against age. Samples rarefied at 5,000 sequences/sample. (B) Average Faith s phylogenetic diversity of oral samples from US and Yanomami subjects plotted against age. Samples rarefied at 1,500 sequences/sample. (C) Average Faith s phylogenetic diversity of skin samples from US and Yanomami subjects plotted against age. Samples rarefied at 1,500 sequences/sample.

0.8 0.6 All samples 10K samples 20K samples 100K samples M 2 0.4 0.2 all_100 all_500 all_1k all_5k 10K_r1K 10K_r5K 10K_r10K 20K_r1K 20K_r5K 20K_r10K 100K_r1K 100K_r5K 100K_r10K Samples/Rarefaction level Figure S10. Procrustes analyses of shotgun metagenomic and 16S rrna data for different subsets of samples. The vertical axis displays Procrustes M 2 between shotgun and 16S rrna data. The horizontal axis presents subsets of samples ( all = all samples; 10K = samples with at least 10,000 reads mapped to IMG genoms; 20K = samples with at least 20,000 reads mapped; 100K = samples with at least 100,000 reads mapped) and rarefaction cutoffs (e.g. all_100 = all samples, rarefied at 100 seqs/sample; 10K_5K = samples with 10K mapped reads, rarefied at 5,000 seqs/sample). A representative Procrustes plot is shown on top for each subset of samples (all samples, samples with >10K mapped reads, samples with >20K mapped reads, samples with >100K mapped reads).

A MG.F3 MG.F5 MG.F6 MG.F9 1 B MG.F3 0.9 0.8 0.30 0.7 MG.F5 MG.F6 0.6 0.5 0.4 0.3 M 2 0.28 0.26 0.2 MG.F9 0.1 0 0.24 MG.O12 MG.O13 MG.O14 MG.O19 MG.O23 MG.O3 MG.O4 MG.O49 MG.O5 MG.O50 MG.O51 MG.O6 MG.O9 MG.O12 MG.O13 MG.O14 MG.O19 MG.O23 MG.O3 MG.O4 MG.O49 MG.O5 MG.O50 MG.O51 MG.O6 MG.O9 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 C Figure S11. Comparison of PICRUSt versus sequenced shotgun metagneomes of fecal and oral samples with at least 10,000 mapped reads. (A) Pearson correlation coefficient between PICRUSt-predicted and sequenced metagenomes for each sample. Size and color of the circles indicate strength of the correlation. (B) Procrustes analysis between PICRUSt-predicted and sequenced metagenomes. 100 rarefied PICRUSTpredicted and shotgun sequenced metagenomes were compared using Procrustes. The M 2 values of the comparisons, indicating goodness of fit (the lower the better), are presented (p < 0.01, Monte Carlo permutation test). (C) Representative Procrustes plot between PICRUSt and shotgun metagenomic tables.

Figure S12. Bacterial genome assembly. (A) Shotgun metagenome assembly statistics per k-mer size. (Top) Number of reads mapped to contigs. (Middle) N50 statistic. (Bottom) Number of contigs. (B) Quality metrics of metagenome assembly. Statistics for cluster quality at different k-mer values (31 to 91) and three thresholds for contig length: All contigs above 500bp (top), all contigs above 200bp (middle), and all contigs above 100bp (bottom). Adjusted Rand = adjusted rand index; F1 measure =2*(precision * recall) / (precision + recall). NMI = Normalized Mutual Information. Rand = rand index. K-mers that resulted in best overall statistics are shaded (k=59, 63, 67 for >500bp contigs; k=59 for >200bp contigs; k=55, 59 for >100bp contigs). (C) Contig clusters of genome assembly. Contig clusters obtained with CONCOCT 0.3.3 using contigs of >500bp (k=59, 63, 67), >200bp (k=59), and >100bp (k=55, 59).